PROJECT SUMMARY/ABSTRACT This proposal seeks support for a translational grant funded continuously by NICHD for the past 26 years. Altered partitioning of fat, independent of obesity, during adolescence carries a high risk for insulin resistance (IR), T2D and Fatty Liver. Over the last decade, we formed ?The Yale Study of Body Fat Patterning in Obese Adolescents? cohort, assessed the role of body fat distribution as a modifier of glucose/insulin metabolism and described a distinct ?metabolic phenotype? typified by a thin superficial layer of abdominal SAT, increased VAT, fatty liver and marked IR . Recently, longitudinal follow-up of this cohort revealed that the High VAT/(VAT+SAT) phenotype is the best predictor of fatty liver in youth. To unravel the cellular mechanisms underlying the inefficient fat storage in abdominal SAT we began, in the current cycle, with Marc Hellerstein to employ the 2H2O labeling method to measure in vivo the dynamic fluxes of triglycerides, De Novo Lipogenesis (DNL) and adipocyte turnover in the abdominal and gluteal SAT . Highlights: Obese adolescent girls with High VAT/(VAT+SAT) display: a- Increased in vivo rates of lipolysis in abdominal and gluteal SAT; b- higher adipocyte turnover; c- the increased lipolytic rates related strongly to Fatty Liver. Building on these findings, the overarching goal of this proposal is: To unravel the underlying molecular mechanisms by which impaired insulin-mediated suppression of lipolysis from two SAT depots occurs and its link with Fatty Liver, in obese adolescents with the unfavorable pattern of abdominal fat distribution. The Specific Aims & Hypotheses (H) are: Aim 1 & H1 - To determine the underlying molecular mechanisms causing the increased in vivo lipolysis flux in the abdominal and gluteal SAT depots of obese with High VAT/(VAT+SAT) compared to BMI, age, and gender matched obese youths with Low VAT/(VAT+SAT). H1: Increased WAT lipolysis in obese youth with High VAT/(VAT+SAT) will be associated with decreased insulin-stimulated insulin receptor kinase (IRK) phosphorylation leading to increased ATGL, CGI-58, PLIN1 and HSL phosphorylation. Aim 2 & H2a- To test whether increased rates in gluconeogenesis (GNG) associated with the excessive WAT lipolysis in the SAT contribute to hepatic IR in obese youths with High VAT/(VAT+SAT) compared to BMI, age, sex matched obese youths with the Low VAT/(VAT+SAT). H2b- Increased rates of WAT lipolysis in obese youth with High VAT/(VAT+SAT) will correlate with increased rates of hepatic GNG, which in turn can be attributed to increased hepatic acetyl-CoA content as reflected by increased rates of ?-OHB turnover. Aim 3 & H3: To determine the mechanisms mediating the temporal development of fatty liver we will test the hypothesis that changes in De Novo lipogenesis (DNL) over time will be associated with changes in fatty liver in obese youths. state-of-the?art Overall Approach: In lean and obese youth recruited for this study we will use a combination of cellular/molecular techniques (G.I Shulman), clamp+isotopes, MRI and in vivo measures of lipid fluxes (S. Caprio & N. Santoro) and adipocyte turnover (2H2O labeling method) (M. Hellerstein) in SAT depots.